1. Field of the Invention
The present invention discloses a process for regioselectively preparing phosphorylated cyclitols, in particular phosphorylated inositols such as myo-inositol 1,4,5-tris(phosphate) and myo-inositol 1,3,4,5-tetrakis (phosphate). Novel cyclitols produced by means of this process are also described.
2. Background of the Invention
Cyclitols are cycloalkanes containing one hydroxyl group on each of three or more ring carbons. The most abundant members of the cyclitol family are the inositols (1,2,3,4,5,6-hexahydroxycyclohexanes) and the most important stereoisomer of this family is myo-inositol which has the 1-, 2-, 3-, and 5-hydroxyl groups on one side of the ring and the 4- and 6-hydroxyl groups on the other. Phosphorylated derivatives of cylitols and inositols, that is, those which have one or more hydroxyl groups converted to phosphate monoesters, are generally referred to, respectively, as cyclitol phosphates or inositol phosphates.
Cellular processes of all animals, including man, depend, at least in part, upon inositol phosphates. Certain inositol phosphates function as "second messengers", that is, molecules which provide the means by which neurotransmitters, growth factors or hormones alter processes inside cells without necessarily penetrating the cells they affect. When the circulating hormone vasopressin binds to receptors on liver cells, for example, it stimulates an increase in intracellular concentrations of D-myo-inositol 1,4,5-tris(phosphate) and D-myo-inositol 1,3,4,5-tetrakis(phosphate). The increased concentrations of these second messengers in turn activates certain enzymatic processes within the cells. Similarly, some growth factors such as platelet derived growth factor (PDGF) cause increased production of inositol phosphates in the cells they affect. Intracellular concentrations of inositol phosphates also appear to play a role in the regulation of cell division and the inflammatory response. Because of the potential medicinal importance of the natural inositol phosphates, and analogs and isomers thereof, considerable research interest in these compounds has been generated. The National Library of Medicine's MEDLINE.TM. lists more than 1400 published papers since 1980 on the subject of inositol phosphates. Recent reviews can be found in Science, 234: 1519 (1986) and Scientific American, 253: 142 (1985).
Studies of inositol phosphates have been hindered by the limited amounts of material which are tediously isolated from natural sources. Practical synthetic routes for preparing significant amounts of these compounds or their analogs or isomers are not currently available. Research efforts would benefit greatly from the availability of adequate quantities of isomerically-pure synthetic materials. An object of the present invention is to provide a synthetic process for the efficient preparation of naturally occurring inositol phosphates and analogs and isomers thereof.
The broad steps utilized in art processes to synthesize phosphorylated cylitols such as inositols are as follows. First, an inositol compound having appropriately protected hydroxyl groups is obtained (Step 1). Next, the free hydroxyl groups in these precursors are converted to phosphate groups (Step 2), and finally, the hydroxyl protecting groups are removed (Step 3). In practice, the second step frequently consists of the following two stages: Step 2a, formation of a phosphorus-oxygen bond between the phosphorus of a "phosphorylating agent" (a compound where the phosphorus is in the +5 oxidation state (P(+5)), and one or more inositol oxygens; and Step 2b, removal of any protecting groups which are on the phosphorylating agent, hereafter referred to as phosphorus protecting groups. The crucial element in the synthesis of these compounds is the phosphorylation step (Step 2a) and the agents which effect it. For a general review of phosphorylation see Slotin, Synthesis, 737 (1977).
Three types of P(+5) phosphorylating agents have generally been employed in Step 2a:
Type I: (RO).sub.2 --P(.dbd.O)--X; PA0 Type II: (RO).sub.2 --P(.dbd.O)--OH or salt thereof; and PA0 Type III: (RNH).sub.2 --P(.dbd.O)--X or RCONH--P(.dbd.O)(OH)(O.sup.-); PA0 a halogen, i.e., F, Cl, Br or I, NR.sub.2.sup.2, where R.sup.2 is aryl or a C.sub.1 -C.sub.15 PA0 OH or a salt thereof; PA0 O, PA0 S, PA0 NH, and PA0 NR.sup.2 ; PA0 aryl, and PA0 a C.sub.1 -C.sub.15 straight chain, branched or cyclic alkyl, PA0 where the alkyl may be internally interrupted with ether oxygen and where aryl or the alkyl may be substituted or unsubstituted with nitro, sulfonyl, halogen ester and ketone groups; provided that
wherein R=phosphorus protecting groups and X=halogen.
The conversion of hydroxyl groups to phosphate monoesters generally has been accomplished by the action of a phosphorylating agent of Type I. One problem with most of the Type I agents is that phosphorylating agents which contain phosphorus(+5) atoms are not sufficiently reactive to efficiently polyphosphorylate partially protected inositols. Monophosphorylation of 1,2,3,5,6-pentabenzylinositol with the classical phosphorylating agent, diphenyl chlorophosphate (Type I, wherein R=C.sub.6 H.sub.5 and X=Cl), for example, affords only a 70% yield of the desired inositol diphenyl monophosphate (Billington et al., J. Chem. Soc., 314 (1987)). Polyphosphorylations, i.e., bis-, tris-, tetrakis-, etc., are difficult to achieve with selectivity, and separation of the many undesired phosphorylated side products from the desired phosphorylated product produced via this reaction scheme presents a difficult task. Forcing conditions, such as higher temperatures are problematic since these phosphorylating agents tend to decompose, as described in Krylova et al., J. Org. Chem. USSR, 16: 277-282 (1980), or effect premature removal of hydroxyl protecting groups, discussed in Krylova et al., Zh. Org. Khim., 42: 702 (1972).
The Type II phosphorylating agents are used in situ after having been converted to a mixed anhydride with an activating agent such as triisopropylbenzenesulfonyl chloride or dicyclohexylcarbodiimide. However, none of these reagents are any more reactive than the Type I reagents, and thus are equally inefficient at phosphorylating inositols. For example, inositol has been converted to a mixture of pentakis- and hexakis(phosphates) by heating in polyphosphoric acid at 120.degree. C., as described by Cosgrove, J. Sci. Food Agric., 17: 550 (1966).
An equally significant disadvantage of the aforementioned Type I and Type II phosphorylating agents is that they have the potential to form cyclic phosphates when the substrate to be phosphorylated contains unprotected vicinal hydroxyl groups, especially cis vicinal hydroxyl groups. For example, when treated with diphenyl chlorophosphate, 1,2:5,6-di-O-iso-propylidiene-(-)-inositol (a trans vicinal diol) and 1,4,5,6-tetra-O-acetyl-myo-inositol (a cis vicinal diol) afford cyclic monophosphates instead of the desired 3,4- and 2,3-bis(phosphates) (Angyal et al., J. Chem. Soc., 4122 (1961)). Although some bis(phosphates) can be prepared from the trans vicinal diols using diphenyl chlorophosphate, precautions must generally be taken with inositol phosphate triesters intermediates because they are exceptionally prone to cyclization onto a neighboring free hydroxyl group. For example, Billington et al., J. Chem. Soc., 314 (1987), find that 2,3,4,5,6-pentabenzyl-myo-inositol-1-(diphenyl phosphate) is deprotected by hydrogenolysis to a mixture of myo-inositol 1- and 2-phosphates. Migration of phosphate occurs because the benzyl ether protecting groups are removed more rapidly than the phosphorus protecting groups. Intermediate phosphate triesters with a free 2-hydroxyl group cyclize and then ring open to form the reported mixture of products.
The above cyclization and migration problems lead to the development of a third type of P(+5) phosphorylating agent, Type III, shown above. In this type of agent, one or two of the phosphorus oxygens are replaced by nitrogens. The phosphorylated products, i.e., inositol phosphoramidates or phosphorodiamidates, produced by this type of agent are apparently not as prone to cyclization as are the phosphorus triesters, probably because nitrogen is a poorer leaving group than oxygen. Angyal et al., Aust. J. Chem., 22: 391-404 (1969) reported obtaining a mixture of tetra- and the desired pentaphosphorylated products when attempting to exhaustively phosphorylate 1-O-benzyl-myo-inositol it with the monotriethylammonium salt of N-benzoyl-phosphoramidic acid in dimethylformamide (DMF) at 140.degree. for 24 h. In this case, the phosphorus protecting groups (including removal of the nitrogen) and pyrophosphate intermediates were hydrolyzed with hydrochloric acid. Krylova, J. Org. Chem USSR, 16: 277 (1980), reports that phosphorylation of 1,2-O-cyclohexylidiene-3,6-di-O-benzyl-myo-inositol with "dianilidophosphoric chloride" afforded the desired 4,5-bis(phosphordiamidite). Dianilidophosphoric chloride appears to be about as reactive as the corresponding oxygen analog diphenyl phosphorochloridate. These nitrogen-containing phosphorus protecting groups can be removed by hydrolysis with aqueous hydrochloric acid or buffered nitrous acid. The former method has the potential to cause migration of phosphate groups and the highest yield reported for the latter method is 25%. These Type III phosphorylation agents, therefore, while offering hope of obtaining polyphosphates from a variety of protected inositols, have a number of unresolved problems associated with their use.
The first total synthesis of inositol 1,4,5-tris(phosphate) was reported by Ozaki et al., Tetrahedron Lett., 27: 3157-3160 (1986). This synthesis illustrates the weaknesses of the best existing technology when applied to a synthetically difficult, biologically important molecule. Ten steps of chemistry were required to prepare (+)-2,3,6-tri- benzylinositol, an appropriate precursor for phosphorylation. Tris(phosphorylation) was effected by means of dianilidochlorophosphate in "ca. 41% yield". The authors noted that "[a]t the present time, phosphorylation and subsequent deblocking reaction[sic,reactions] [using isoamyl nitrite] are not satisfactory". No overall yield of final product was given. Gigg et al., Carbohydrate Res., 140: cl (1985)) also reported a long synthesis of the same tribenzylinositol, but they have apparently been unable to obtain the desired tris(phosphate) from this precursor.
In conclusion, the regioselective synthesis of many inositol phosphates by means of the existing P(+5) technology is a long, risky and inefficient process.
Over the past decade, phosphorylation with phosphorus(+3) (P(+3)) phosphorylating agents (also known as phosphitylating agents) has revolutionized the field of oligonucleotide synthesis. First chlorophosphite diesters, developed by Letsinger et al., J. Am. Chem. Soc., 97: 3278 (1975), and then more recently phosphoramidites, developed by Beaucage et al., Tetrahedron Lett., 22: 1859 (1981), have been the reagents of choice for the construction of phosphorus diester, as opposed to monoester, bonds in oligonucleotides. The use of phosphorus(+3) reagents for the construction of phosphorus diesters requires that two key hydroxyl groups become attached to a monoprotected phosphitylating agent. Adapting phosphorus(+3) methodology to the preparation of phosphorus monoesters requires that one key hydroxyl group become attached to a phosphitylating agent with two protecting groups.
There are scattered reports in the literature on methods for preparing phosphate monoesters by means of phosphitylation agents. Many of these reports, however, involve the use of phosphitylating agents designed for making phosphate diesters and one would anticipate that these agents would produce cyclic phosphite esters from many inositol substrates. Bannwarth et al., Helv. Chim. Acta, 70: 175-186 (1987), report a comprehensive study on appropriate phosphitylating agents for making phosphate monoesters. Kozlova et al., J. Gen. Chem. USSR, 39: 2403-2406 (1969), disclose the only example of phosphitylation of a cyclitol derivative. These authors, however, were interested in the preparation of phosphate diesters of inositol, that is, phospholipids. Consequently, the phosphitylating agent they chose to use (the mixed anhydride from O-benzylphosphorous acid and O,O-diphenylphosphoric acid) is one which might be anticipated to produce cyclic phosphite esters with many of the inositol substrates of interest to this invention. Since all of the other hydroxyl groups in the inositol substrate they used were protected, they observed no cyclization and obtained an inositol (mono-) phosphite diester as the major product. No phosphite triesters of inositol are known.
The present invention centers around the creation of a superior process that uses phosphitylation as a key step in the preparation of cyclitol phosphates, particularly inositol phosphates. Prior to the studies disclosed herein, no one had used phosphitylation to prepare cyclitol phosphate monoesters nor had anyone used phosphitylation to attach more than one phosphorus to cyclitols. The general usefulness of phosphitylation for the preparation of poly(phosphates) from starting materials with more than one hydroxyl group did not appear to have been established. In particular, there was no evidence demonstrating that changing to methods based on phosphitylation would alleviate the side reactions which interfere with more conventional phosphorylating agents, i.e., incomplete poly(phosphorylation), cyclization, and phosphate migration.
Although the phosphitylating agents used in the present process have been employed in other systems, they have never been applied to the problem of producing inositol phosphates and other cyclitol phosphates, and indeed, their ability to effectively work in the present system and avoid many of the problems prevalent in the art processes is quite surprising.